Name: CALB

Base Pairs: 1029 bp

Origin:

The gene encoding Candida antarctica Lipase B (CALB) originates from the yeast Candida antarctica. We performed codon optimization on this gene sequence and commercially synthesized for high-level expression in Pichia pastoris.

Properties:

CALB is a serine hydrolase belonging to the α/β-hydrolase fold family. This enzyme exhibits remarkable catalytic versatility, demonstrating activity in hydrolysis, esterification, transesterification, and amidation reactions. Its distinctive properties include high thermostability (up to 60°C), broad substrate specificity, and exceptional enantioselectivity, particularly for sec-alcohols and esters.

Usage and Biology:

CALB is extensively used in industrial biocatalysis for the production of pharmaceuticals, agrochemicals, and fine chemicals, leveraging its stereoselectivity for chiral synthesis. Additional applications include biodiesel production, flavor compound synthesis, and polymer chemistry. CALB operates through a classical catalytic triad (Ser-His-Asp) mechanism, characterized by a unique, narrow substrate-binding tunnel that contributes to its stereoselectivity. The enzyme's stability is enhanced by its rigid structure and glycosylation pattern, while its ability to function in both aqueous and non-conventional media makes it particularly valuable for industrial bioprocesses [1].

Name: GGL

Base Pairs: 1692 bp

Origin:

The GGL gene originates from the yeast strain Galactomyces geotrichum Y05. We performed codon optimization on this gene sequence and commercially synthesized for high-level expression in Pichia pastoris.

Properties:

GGL encodes an extracellular glycoprotein belonging to the glycoside hydrolase family. This enzyme exhibits broad substrate specificity, demonstrating activity on β-linked glucosyl residues and mixed-linkage polysaccharides. It functions optimally under acidic to neutral pH conditions and shows notable stability in the presence of organic solvents, making it suitable for industrial biocatalytic processes.

Usage and Biology:

GGL is primarily employed in biorefinery applications for the degradation of plant biomass, particularly in the saccharification of hemicellulose components for biofuel production. Additional applications include its use in animal feed supplementation to improve digestibility and in food processing for modifying textural properties. GGL participates in carbon cycle metabolism by hydrolyzing complex polysaccharides into assimilable sugars. The enzyme operates through a retaining mechanism involving a catalytic glutamic acid residue, with its glycoprotein nature enhancing stability in extracellular environments. This enzymatic activity enables the host organism to utilize plant-derived materials as nutrient sources in natural habitats [2].

Name: LipZ01

Base Pairs: 1425 bp

Origin:

The LipZ01 gene was identified through functional screening of a soil-derived metagenomic library. We performed codon optimization on this gene sequence and commercially synthesized for high-level expression in Pichia pastoris.

Properties:

LipZ01 encodes a novel serine hydrolase belonging to the lipase family. This enzyme demonstrates several distinctive characteristics: maintained activity across a broad temperature range, exceptional stability under alkaline conditions, high compatibility with detergent components, specific preference for long-chain triglycerides, and superior catalytic efficiency in transesterification reactions compared to conventional lipases.

Usage and Biology:

The enzyme is primarily employed in industrial applications including biodiesel production through transesterification, detergent formulations for lipid stain removal, and food processing for flavor modification. LipZ01 operates through a classic catalytic triad mechanism, with its unique structural features enabling remarkable solvent tolerance and interfacial activation at lipid-water interfaces. The enzyme's ability to maintain conformation flexibility under harsh conditions contributes to its outstanding performance in both aqueous and non-aqueous reaction systems, particularly in organic synthesis applications requiring high conversion rates [3].

After the company synthesized three lipases that had been optimized for codons, we inserted them into a P. pastoris expression vector pPICZαA, which includes the methanol-inducible AOX1 promoter (PAOX1), the α-factor secretion signal, and a C-terminal His-tag, followed by the AOX1 terminator. The Zeocin resistance gene enables selection in transformants (Figure 1A). After successfully obtaining the plasmid, we linearized it and transferred it into P. pastoris. These results collectively validate the successful genomic integration of all three lipase expression cassettes into P. pastoris, providing a solid foundation for subsequent protein expression studies.

Figure 1 The construction result of Pichia pastoris engineered with the lipase gene.

(A) Plasmid design diagram. (B) Transformation plates. (C) Yeast colony results.

Fermentation and quantitative analysis revealed that only CALB was efficiently secreted into the culture supernatant, achieving a concentration of 68.21 ± 4.94 µg/mL and an enzymatic activity of 28.59 ± 0.37 U/mL. In contrast, the secretion levels of GGL (4.89 µg/mL) and LipZ01 (1.61 µg/mL) were negligible. Given these results, subsequent optimization efforts will focus exclusively on CALB, with the goal of further enhancing its expression and secretion through systematic engineering of promoters and signal peptides.

Figure 2 The results of (A-B) lipase secretion and (C) enzyme activity.

Name: AOX713 promoter

Base Pairs: 757 bp

Origin:

The AOX713 promoter is an engineered variant derived from the native alcohol oxidase 1 (AOX1) promoter of Pichia pastoris. This synthetic promoter was developed through rational design and screening strategies to enhance regulatory control and expression efficiency in this yeast expression system.

Properties:

AOX713 is a strong, inducible promoter that exhibits tighter regulatory control and reduced basal expression compared to the wild-type AOX1 promoter. It maintains strict methanol responsiveness while demonstrating improved transcriptional activity under induction conditions. The promoter shows compatibility with high-cell-density fermentations and can drive high-level expression of heterologous proteins.

Usage and Biology:

AOX713 promoter is primarily used for controlled recombinant protein production in P. pastoris expression systems, particularly for pharmaceuticals, industrial enzymes, and biotherapeutics requiring precise expression timing. AOX713 promoter functions through a modified regulatory mechanism where transcription factors specifically recognize methanol as an inducer, activating the methanol metabolism pathway. The engineered elements in AOX713 enhance promoter strength by optimizing transcription factor binding sites while maintaining the native pathway's induction specificity, enabling more efficient protein production with reduced metabolic burden on the host cells [4].

Name: FDH1 promoter

Base Pairs: 1008 bp

Origin:

The FDH1 promoter is an endogenous methanol-inducible promoter derived from the methylotrophic yeast Pichia pastoris. It is obtained by cloning the regulatory region upstream of the formate dehydrogenase 1 (FDH1) gene, often with sequence optimization to enhance its transcriptional activity.

Properties:

The FDH1 promoter is an endogenous methanol-inducible promoter derived from the methylotrophic yeast Pichia pastoris. This gene encodes formate dehydrogenase, a key enzyme in methanol metabolism. FDH1 promoter is strongly induced by methanol and inhibited by other carbon sources such as glucose or glycerol, making it one of the tools for efficient heterologous gene expression in yeast cell factories.

Usage and Biology:

FDH1 promoter is widely used for controlled heterologous protein expression in P. pastoris, especially for producing toxic proteins or enzymes requiring precise induction timing. The FDH1 promoter is activated by methanol through the same regulatory pathway as AOX1 but is subject to stronger carbon catabolite repression. Its induction relies on the transcription factors Mit1 and Mxr1, which bind to specific motifs in the promoter region upon methanol sensing, while being strongly repressed by glucose/glycerol via the Mig1 repressor complex, ensuring tight regulatory control [5].

Name: FLD1 promoter

Base Pairs: 597 bp

Origin:

The FLD1 promoter is an endogenous bidirectional promoter derived from the methylotrophic yeast Pichia pastoris. It is obtained from the genomic region upstream of the formaldehyde dehydrogenase 1 (FLD1) gene, which plays a central role in methanol and methylamine metabolism.

Properties:

The FLD1 promoter is a uniquely versatile inducible promoter characterized by its dual induction mechanism. It can be strongly activated either by methanol as a sole carbon source (with ammonium sulfate as nitrogen source) or by methylamine as a sole nitrogen source (with glucose as carbon source). The promoter exhibits induction strength comparable to the AOX1 promoter while offering greater flexibility in fermentation design.

Usage and Biology:

This promoter is particularly valuable for expressing heterologous proteins under conditions where methanol usage is restricted or when utilizing alternative induction strategies to reduce metabolic stress. FLD1 promoter is regulated by two distinct pathways: methanol induction occurs through the Mxr1 transcription factor activating the methanol metabolism pathway, while methylamine induction operates via the nitrogen limitation response pathway involving transcription factor Amt1. This dual regulation allows the promoter to respond to distinct metabolic signals, making it a robust and flexible tool for metabolic engineering and high-level protein production [6].

Achieving efficient expression of lipases in P. pastoris requires careful selection and optimization of promoters, as they directly govern the efficiency and intensity of target gene transcription initiation [7]. The pPIC9K vector is a commonly used tool in the P. pastoris expression system, which incorporates the potent methanol-inducible AOX1 promoter, and utilizes the α-factor signal peptide to direct the secretory expression of recombinant proteins. This vector is marked for prokaryotic screening in E. coli with ampicillin (Amp) and kanamycin (Kan) resistance, while screening in P. pastoris utilizes the HIS4 marker. (Figure 3A). After successfully obtaining the plasmid, we linearized it and transferred it into P. pastoris. These results collectively validate the successful genomic integration of all three lipase expression cassettes (Figure 3B-C).

Figure 3 The construction results of Pichia pastoris.

(A) Plasmid design diagram. (B) Transformation plates. (C) Yeast colony results.

The SDS-PAGE results confirmed successful CALB secretion, with distinct protein bands visible in all experimental lanes but absent in the negative control (Figure 4A). Quantitative analysis revealed significant differences in secretion levels, with FDH1 promoter yielding the highest CALB concentration (93.82 ± 4.16 μg/mL), followed by AOX713 (86.04 ± 8.53 μg/mL), AOX1 (68.21 ± 4.94 μg/mL), and FLD1 (61.25 ± 3.32 μg/mL) (Figure 4B). Enzymatic activity assays demonstrated a similar trend, where FDH1 promoter achieved the highest activity (31.68 U/mL), significantly outperforming other promoters (Figure 4C). These results collectively establish FDH1 as the most effective promoter for CALB expression under the tested conditions.

Figure 4 The results of (A-B) lipase secretion and (C) enzyme activity.

Name: 0030-α-factor SP

Base Pairs: 267 bp

Origin:

The 0030-α hybrid signal peptide was created by fusing the pre-sequence of the 0030 signal peptide with the pro-sequence of the Saccharomyces cerevisiae α-factor, forming a chimeric secretion signal.

Properties:

This chimeric signal peptide combines the efficient endoplasmic reticulum (ER) export signaling capability of the α-factor pro-sequence with enhanced transmembrane translocation efficiency conferred by the engineered 0030 pre-sequence. The hybrid maintains the necessary structural features for recognition by the Sec61 translocon complex while improving the initiation and efficiency of the protein translocation process across the ER membrane.

Usage and Biology:

The 0030-α hybrid is primarily employed to enhance recombinant protein secretion in yeast expression platforms such as Pichia pastoris. The 0030 pre-sequence facilitates superior initial recognition and translocation through the translocon complex, while the α-factor pro-sequence ensures efficient ER export and engagement with the vesicular transport machinery. This dual optimization significantly increases secretion yields of target proteins while reducing intracellular accumulation, thereby streamlining downstream purification processes in biomanufacturing applications [8].

Name: SWP1-α-factor SP

Base Pairs: 252 bp

Origin:

The SP4-α signal peptide is an engineered hybrid created by fusing the N-terminal 18-amino acid sequence (MKLFFVGIVTGLLTLLVSC) derived from the SWP1 protein with the pro-sequence of the Saccharomyces cerevisiae α-factor signal peptide, forming a novel chimeric secretion signal.

Properties:

This hybrid signal peptide combines the strong hydrophobic transmembrane domain of the SWP1-derived sequence with the efficient endoplasmic reticulum export signaling capability of the α-factor pro-region. Experimental validation has demonstrated its ability to enhance secretion efficiency of various recombinant proteins by 3-5 fold compared to conventional signal peptides, while maintaining proper recognition by the cellular secretion machinery.

Usage and Biology:

The SP4-α hybrid is primarily employed to significantly improve recombinant protein secretion in yeast expression systems. SWP1-derived N-terminal region facilitates efficient initial targeting and translocation through the Sec61 translocon complex via its optimized hydrophobic core, while the α-factor pro-sequence ensures proper folding in the endoplasmic reticulum and subsequent vesicular transport through the Golgi apparatus. This synergistic action results in dramatically increased extracellular protein yields while reducing endoplasmic reticulum stress and intracellular protein aggregation, making it particularly valuable for industrial production of therapeutic proteins and industrial enzymes [9].

Name: KRE1-α-factor SP

Base Pairs: 252 bp

Origin:

The KRE1-α signal peptide is a hybrid secretion signal engineered by fusing the N-terminal 18-amino acid sequence (MKLFFVGIVTTLLTLLVSC) from the Pichia pastoris cell wall synthesis-related protein KRE1 with the pro-sequence of the Saccharomyces cerevisiae α-factor signal peptide.

Properties:

This engineered hybrid combines the strong hydrophobic transmembrane domain of KRE1 with the efficient endoplasmic reticulum export signaling capability of the α-factor pro-region. Experimental validation confirms its exceptional performance in enhancing secretion efficiency, achieving over fivefold improvement in extracellular production of multiple recombinant proteins compared to conventional signal peptides, while maintaining full compatibility with the host cell's protein processing machinery..

Usage and Biology:

The KRE1-α hybrid is primarily employed to dramatically enhance recombinant protein secretion in P. pastoris expression systems. KRE1-derived N-terminal region mediates efficient initial membrane targeting and Sec61 translocon-mediated translocation through its optimized hydrophobic characteristics, while the α-factor pro-sequence ensures proper protein folding in the endoplasmic reticulum and facilitates subsequent vesicular transport through the secretory pathway. This synergistic mechanism significantly boosts extracellular protein yields while simultaneously reducing endoplasmic reticulum stress and intracellular protein aggregation, making it particularly valuable for industrial bioprocessing of therapeutic proteins and challenging-to-express enzymes [9].

To optimize the secretion efficiency of lipase in P. pastoris, this study replaced the original α-factor signal peptide of the pPIC9K vector with engineered ones. We introduced three highly efficient secretion signal peptides that have been verified in the literature: 0030α-factor, SWP1-α-factor, and KRE1-α-factor, which retained the AOX1 promoter on the pPIC9K vector (Figure 5A). After successfully obtaining the plasmid, we linearized it and transferred it into P. pastoris. These results collectively validate the successful genomic integration of all three lipase expression cassettes (Figure 5B-C).

Figure 5 The construction results of Pichia pastoris.

(A) Plasmid design diagram. (B) Transformation plates. (C) Yeast colony results.

Fermentation of three engineered P. pastoris strains revealed distinct 43.8 kDa bands on SDS-PAGE, confirming CALB secretion. Quantitative analysis showed the 0030-α signal peptide yielded the highest CALB concentration (90.76 ± 3.65 μg/mL), significantly surpassing the standard α-factor (68.21 ± 4.94 μg/mL, ​​P ≤ 0.001). SWP1-α also enhanced secretion (69.62 ± 5.13 μg/mL), while KRE1-α performed poorly (56.28 ± 1.79 μg/mL) (Figure 6A-B). Enzymatic activity assays confirmed 0030-α-CALB had the highest activity (31.25 U/mL), significantly exceeding the α-factor control (28.59 U/mL, P ≤ 0.05). SWP1-α and KRE1-α variants showed comparable activity to the control (ns) (Figure 6C). These results demonstrate that signal peptide engineering, particularly using 0030-α, significantly enhances both CALB secretion and catalytic efficiency in P. pastoris.

Figure 6 The results of (A-B) lipase secretion and (C) enzyme activity.